Acetylcholinesterase (AChE) is an important enzyme responsible for the cleavage of acetylcholine. Studies of the activity of this enzyme use an artificial substrate, acetylthiocholine, because a product of its catalys...Acetylcholinesterase (AChE) is an important enzyme responsible for the cleavage of acetylcholine. Studies of the activity of this enzyme use an artificial substrate, acetylthiocholine, because a product of its catalysis, thiocholine, readily generates a light absorbing product upon reaction with Elman’s reagent 5,5’-dithiobis-(2-nitrobenzoic acid (DTNB). The hydrolysis of acetylcholine cannot be assayed with this method. The isothermal titration calorimetry can assay the hydrolysis of both substrates, without requiring additional reagents other than the enzyme and the substrate. To compare kinetic values obtained in the hydrolysis of acetylcholine (ACh) and acetylthiocholine (ATCh), with carbaryl acting as inhibitor, a calorimetric technique was used to evaluate kinetic properties of the two reactions. This method can show the hydrolysis of both substrates by the heat exchange that occurs during catalysis. In addition, it allowed the assessment of the AChE inhibition by carbaryl, a common insecticide. The results show a similarity between values obtained with both substrates, which are slightly higher for acetylcholine, the enzyme natural substrate. Enzymatic parameters values from ATCh and ACh were similar to each other and inhibitory constants using carbaryl were also similar, displaying that any approach to ACh is feasible using ATCh. The results obtained from ITC show the precision achieved by the calorimetric method.展开更多
This study describes the measurement of bio-electrical signals caused by enzymatic inhibition of acetylcholinesterase (AChE) for the detection of organophosphorous and carbamate pesticides which are the strong inhibit...This study describes the measurement of bio-electrical signals caused by enzymatic inhibition of acetylcholinesterase (AChE) for the detection of organophosphorous and carbamate pesticides which are the strong inhibitors of AChE and prevents its normal function of the rapid removal of acetylcholine (Ach). Biosensor Toxicity Analyzer (BTA) was used for the testing and enzyme activity was determined by acetylthiocholine chloride (ATCCl) as enzyme substrate. The monitoring of changes in bio-electrical signals caused by the interaction of biological substances and residues were evaluated. Two samples of cotton were analyzed. Cryogenic homogenization was carried out for sample pretreatment and Soxhlet extraction method (SOX) was used for extraction. The resulted extracts were concentrated and then injected in the BTA. The method shows reasonable results and can successfully be utilized for the detection of residual pesticides on different types of cotton.展开更多
基金supported by Brazilian Ministry of Health(n.17217.9850001/12-025).
文摘Acetylcholinesterase (AChE) is an important enzyme responsible for the cleavage of acetylcholine. Studies of the activity of this enzyme use an artificial substrate, acetylthiocholine, because a product of its catalysis, thiocholine, readily generates a light absorbing product upon reaction with Elman’s reagent 5,5’-dithiobis-(2-nitrobenzoic acid (DTNB). The hydrolysis of acetylcholine cannot be assayed with this method. The isothermal titration calorimetry can assay the hydrolysis of both substrates, without requiring additional reagents other than the enzyme and the substrate. To compare kinetic values obtained in the hydrolysis of acetylcholine (ACh) and acetylthiocholine (ATCh), with carbaryl acting as inhibitor, a calorimetric technique was used to evaluate kinetic properties of the two reactions. This method can show the hydrolysis of both substrates by the heat exchange that occurs during catalysis. In addition, it allowed the assessment of the AChE inhibition by carbaryl, a common insecticide. The results show a similarity between values obtained with both substrates, which are slightly higher for acetylcholine, the enzyme natural substrate. Enzymatic parameters values from ATCh and ACh were similar to each other and inhibitory constants using carbaryl were also similar, displaying that any approach to ACh is feasible using ATCh. The results obtained from ITC show the precision achieved by the calorimetric method.
文摘This study describes the measurement of bio-electrical signals caused by enzymatic inhibition of acetylcholinesterase (AChE) for the detection of organophosphorous and carbamate pesticides which are the strong inhibitors of AChE and prevents its normal function of the rapid removal of acetylcholine (Ach). Biosensor Toxicity Analyzer (BTA) was used for the testing and enzyme activity was determined by acetylthiocholine chloride (ATCCl) as enzyme substrate. The monitoring of changes in bio-electrical signals caused by the interaction of biological substances and residues were evaluated. Two samples of cotton were analyzed. Cryogenic homogenization was carried out for sample pretreatment and Soxhlet extraction method (SOX) was used for extraction. The resulted extracts were concentrated and then injected in the BTA. The method shows reasonable results and can successfully be utilized for the detection of residual pesticides on different types of cotton.
